Polymerase template switching is an essential mechanism in coronaviruses (CoVs) enabling both subgenomic RNA synthesis and increasing genomic diversity via recombination. Despite its importance, the CoV polymerase template-switching molecular mechanism remains unclear. Using magnetic tweezers, we show that the CoV nonstructural protein (nsp) 13-helicase drives intramolecular polymerase template switching, followed by copy-back RNA synthesis. This activity requires nsp13-helicase adenosine triphosphatase activity and a duplex RNA downstream of the CoV polymerase. Remdesivir and molnupiravir are antiviral nucleotide analogs reported to stall the viral polymerase and induce mutations in genome, respectively. Unexpectedly, we show that their incorporation in the nascent strand increases copy-back RNA synthesis in vitro and decreases recombination events in infected cells. We propose a mechanism of action where these analogs' incorporation traps replication complex in a recombination intermediate, preventing viral RNA utilization. Our study highlights the importance of investigating nucleotide analog mechanisms in replication complexes beyond the polymerase.

The SARS-CoV-2 pandemic has underscored the urgent need for broad-spectrum antivirals in pandemic preparedness efforts. Nucleoside analogs targeting viral polymerases are often considered in this context. Here, we employ ensemble biochemical assays and single-molecule magnetic tweezers to characterize the detailed mechanism of action of the adenosine analog CMX521 (developed through Phase 1 clinical studies), a broad-spectrum antiviral against caliciviruses and coronaviruses, against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The triphosphate form of CMX521 is efficiently incorporated by RdRp, even against saturating ATP concentrations. Analog incorporation induces only a brief pause in nascent RNA synthesis. When embedded in the template strand, CMX521 causes the polymerase to stall ~9 s on average due to impaired uridine opposite incorporation. Multiple CMX521 residues in the template strand completely inhibit polymerase elongation. When the coronavirus polymerase is associated with the viral helicase, CMX521 strongly promotes copy-back RNA synthesis suggesting a second inhibitory mechanism for CMX521. Collectively, our findings establish a two-pronged mode of coronavirus polymerase inhibition by CMX521.

Positive-sense RNA ((+)RNA) viruses often encode helicases presumed to support replication. Their precise role remains unresolved, though, especially in coronaviruses (CoVs), where the helicase translocates in the opposite direction to the polymerase. Using high-throughput single-molecule magnetic tweezers, we show that the coronavirus helicase enhances RNA synthesis through duplex RNA by 10-fold, forming a directional complex with the viral polymerase. Despite opposing polarity, the helicase coordinates elongation by engaging with the non-template strand. A detailed kinetic model derived from large datasets reveals distinct dynamic states, including fast-bursting and slow, backtracking-prone modes, which are governed by helicase engagement. These results uncover an active coupling mechanism that modulates replication dynamics and provide a mechanistic basis for continuous versus discontinuous RNA synthesis in coronaviruses. Our findings establish the viral helicase as a central regulator of RNA replication.

Cells must limit RNA-RNA interactions to avoid irreversible RNA entanglement. Cells may prevent deleterious RNA-RNA interactions by genome organization to avoid complementarity however, RNA viruses generate long, perfectly complementary antisense RNA during replication. How do viral RNAs avoid irreversible entanglement? One possibility is RNA sequestration into biomolecular condensates. To test this, we reconstituted critical SARS-CoV-2 RNA-RNA interactions in Nucleocapsid condensates. We observed that RNAs with low propensity RNA-RNA interactions resulted in more round, liquid-like condensates while those with high sequence complementarity resulted in more heterogeneous networked morphology independent of RNA structure stability. Residue-resolution molecular simulations and direct sequencing-based detection of RNA-RNA interactions support that these properties arise from degree of trans RNA contacts. We propose that extensive RNA-RNA interactions in cell and viral replication are controlled via a combination of genome organization, timing, RNA sequence content, RNA production ratios, and emergent biomolecular condensate material properties.

Coronaviruses (CoVs) encode 16 nonstructural proteins (nsps), most of which form the replication-transcription complex (RTC). The RTC contains a core composed of one nsp12 RNA-dependent RNA polymerase (RdRp), two nsp8s, and one nsp7. The core RTC recruits other nsps to synthesize all viral RNAs within the infected cell. While essential for viral replication, the mechanism by which the core RTC assembles into a processive polymerase remains poorly understood. We show that the core RTC preferentially assembles by first having nsp12-polymerase bind to the RNA template, followed by the subsequent association of nsp7 and nsp8. Once assembled on the RNA template, the core RTC requires hundreds of seconds to undergo a conformational change that enables processive elongation. In the absence of RNA, the (apo-)RTC requires several hours to adopt its elongation-competent conformation. We propose that this obligatory activation step facilitates the recruitment of additional nsps essential for efficient viral RNA synthesis and may represent a promising target for therapeutic interventions.

Polymerase template switching is an essential mechanism in coronaviruses (CoVs) that enables both sub-genomic (sg) RNA synthesis and increases genomic diversity via RNA recombination. Despite its importance, the molecular mechanism of CoV polymerase template switching remains unclear. Using magnetic tweezers, we show that the CoV non-structural protein (nsp) 13-helicase drives polymerase template switching, followed by copy-back RNA synthesis. This activity requires nsp13-helicase ATPase activity and a duplex RNA downstream of the CoV polymerase. This novel function of nsp13-helicase is targeted by the nucleotide analogs remdesivir and molnupiravir, whose incorporation in the nascent strand increases CoV polymerase template switching probability, leading to defective RNA production. We propose a novel mechanism of action where incorporation of these analogs dramatically reduces full length genome copy number by stimulating polymerase template switching. Our study further demonstrates nsp13-helicase's central role in CoV replication and how this enzyme function can be indirectly targeted by analogs.

AbstractG-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5′ single-stranded DNA (ssDNA) segment. Unlike the Thermus oshimai Pif1-G4 structure, in which the 1B and 2B domains confer G4 recognition, ScPif1 recognizes G4 mainly through the wedge region in the 1A domain that contacts the 5′ most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases and beyond.

Pif1 helicase functions in both the nucleus and mitochondria. Pif1 tightly couples ATP hydrolysis, single-stranded DNA translocation, and duplex DNA unwinding. We investigated two Pif1 variants (F723A and T464A) that have each lost one site of interaction of the protein with the DNA substrate. Both variants exhibit minor reductions in affinity for DNA and ATP hydrolysis but have impaired DNA unwinding activity. However, these variants translocate on single-stranded DNA faster than the wildtype enzyme and can slide on the DNA substrate in an ATP-independent manner. This suggests they have lost their grip on the DNA, interfering with coupling ATP hydrolysis to translocation and unwinding. Yeast expressing these variants have increased gross chromosomal rearrangements, increased telomere length, and can overcome the lethality of dna2Δ, similar to phenotypes of yeast lacking Pif1. However, unlike pif1Δ mutants, they are viable on glycerol containing media and maintain similar mitochondrial DNA copy numbers as Pif1 wildtype. Overall, our data indicate that a tight grip of the trailing edge of the Pif1 enzyme on the DNA couples ATP hydrolysis to DNA translocation and DNA unwinding. This tight grip appears to be essential for the Pif1 nuclear functions tested but is dispensable for mitochondrial respiratory growth.

RNA viruses cause many routine illnesses, such as the common cold and the flu. Recently, more deadly diseases have emerged from this family of viruses. The hepatitis C virus has had a devastating impact worldwide. Despite the cures developed in the U.S. and Europe, economically disadvantaged countries remain afflicted by HCV infection due to the high cost of these medications. More recently, COVID-19 has swept across the world, killing millions and disrupting economies and lifestyles; the virus responsible for this pandemic is a coronavirus. Our understanding of HCV and SARS CoV-2 replication is still in its infancy. Helicases play a critical role in the replication, transcription and translation of viruses. These key enzymes need extensive study not only as an essential player in the viral lifecycle, but also as targets for antiviral therapeutics. In this review, we highlight the current knowledge for RNA helicases of high importance to human health.

RNA helicases are responsible for virtually all of RNA metabolism. Viral and bacterial pathogens typically encode their own RNA helicases. Hence, this family of enzymes is increasingly recognized as potential targets for treatment of a variety of diseases. However, the conserved structural similarities among helicase families present an obstacle to the idea of developing specific inhibitors. In order to identify potential modulators of RNA helicase activity, rapid screening approaches are needed. This has been accomplished by optimizing and adapting standard helicase assays to function in high-throughput modalities. These optimized assays have enabled the application of rapid screening approaches to be applied toward discovering helicase inhibitors. This chapter provides detailed protocols for utilizing a medium to high-throughput approach for inhibitor discovery.

G-Quadruplexes are non-B form DNA structures present at regulatory regions in the genome, such as promoters of proto-oncogenes and telomeres. The prominence in such sites suggests G-quadruplexes serve an important regulatory role in the cell. Indeed, oxidized G-quadruplexes found at regulatory sites are regarded as epigenetic elements and are associated with an interlinking of DNA repair and transcription. PARP-1 binds damaged DNA and non-B form DNA, where it covalently modifies repair enzymes or chromatin-associated proteins respectively with poly(ADP-ribose) (PAR). PAR serves as a signal in regulation of transcription, chromatin remodeling, and DNA repair. PARP-1 is known to bind G-quadruplexes with stimulation of enzymatic activity. We show that PARP-1 binds several G-quadruplex structures with nanomolar affinities, but only a subset promote PARP-1 activity. The G-quadruplex forming sequence found in the proto-oncogene c-KIT promoter stimulates enzymatic activity of PARP-1. The loop-forming characteristics of the c-KIT G-quadruplex sequence regulate PARP-1 catalytic activity, whereas eliminating these loop features reduces PARP-1 activity. Oxidized G-quadruplexes that have been suggested to form unique, looped structures stimulate PARP-1 activity. Our results support a functional interaction between PARP-1 and G-quadruplexes. PARP-1 enzymatic activation by G-quadruplexes is dependent on the loop features and the presence of oxidative damage.

G-quadruplex DNA interacts with the N-terminal intrinsically disordered domain of the DEAD-box helicase Ded1p, diminishing RNA unwinding activity but enhancing liquid-liquid phase separation of Ded1p in vitro and in cells. The data highlight multifaceted effects of quadruplex DNA on an enzyme with intrinsically disordered domains.

Helicases are molecular motors with many activities. They use the energy from ATP hydrolysis to unwind double-stranded nucleic acids while translocating on the single-stranded DNA. In addition to unwinding, many helicases are able to remove proteins from nucleic acids. Bacteriophage T4 Dda is able to displace a variety of DNA binding proteins and streptavidin bound to biotinylated oligonucleotides. We have identified a subdomain of Dda that when deleted, results in a protein variant that has nearly wild type activity for unwinding double-stranded DNA but exhibits greatly reduced streptavidin displacement activity. Interestingly, this domain has little effect on displacement of either gp32 or BamHI bound to DNA but does affect displacement of trp repressor from DNA. With this variant, we have identified residues which enhance displacement of some proteins from DNA.

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 μmol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% ± 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca2+ concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 μmol/l) maximally reduced volumetric lymph flow to 34% ± 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% ± 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT: Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications.

Guanine‐rich sequences can interact by Hoogsteen‐hydrogen bonding to form planar quartets that stack into four‐stranded structures known as G‐Quadruplexes (G4DNA). G4DNA is non‐random, overrepresented at key regulatory regions of the cell such as promoters of proto‐oncogenes and telomeres. The overrepresentation in such sites suggests G4DNA serves a significant role in the cell. Poly(ADP‐ribose) polymerase 1 (PARP‐1) is an enzyme that mediates production of polymers of ADP‐ribose units (PAR) that serve as a signal for important cellular processes. PARP‐1 plays a role in the regulation of gene expression, transcription, chromatin remodeling, and DNA repair. Interestingly, PARP‐1 interacts with G4DNA. We show that PARP‐1 binds several G4DNA structures with tight affinity. We also confirm previous evidence revealing that PARP‐1 is enzymatically activated by c‐KIT G4DNA in a DNA structure‐specific manner. In addition, our activity assays reveal that while PARP‐1 binds various G4DNA structures only a subset promote PARP‐1‐mediated PARylation. We show that PARP‐1 enzymatic activation is regulated by G4DNA loop length where longer loops, at least >1 nucleotide, confer higher PARP‐1 activation while shorter loops reduce PARP‐1 activation. Our approach supports a PARP‐1 and G4DNA interaction but emphasizes PARP‐1's catalytic activity is G4DNA loop length‐dependent. Support or Funding Information National Institutes of Health Grant R35 GM122601 (to K.D.R.) UAMS Initiative for Maximizing Student Development Program (IMSD) Grant R25 GM083247 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

We identified 29 G-quadruplex binding proteins by affinity purification and quantitative LC-MS/MS. We demonstrated that the DEAD-box RNA helicases Dbp2, Ded1 and Mss116 preferentially bind to G-quadruplex nucleic acids in vitro and destabilize RNA quadruplexes, suggesting new potential roles for these helicases in disruption of quadruplex structures in RNA.

The hepatitis C virus (HCV) represents a substantial threat to human health worldwide. The virus expresses a dual-function protein, NS3 having both protease and RNA helicase activities that are essential for productive viral replication and sustained infections. While viral protease and polymerase inhibitors have shown great successes in treating chronic HCV infections, drugs that specifically target the helicase activity have not advanced. A robust and quantitative 96-well plate-based fluorescent DNA unwinding assay was used to screen a class of indole thio-barbituric acid (ITBA) analogs using the full-length, recombinant HCV NS3, and identified three naphthoyl-containing analogs that efficiently inhibited NS3 helicase activity in a dose-dependent manner, with observed IC50 values of 21-24 mu M. Standard gel electrophoresis helicase assays using radiolabeled duplex DNA and RNA NS3 substrates confirmed the inhibition of NS3 unwinding activity. Subsequent anisotropy measurements demonstrated that the candidate compounds did not disrupt NS3 binding to nucleic acids. Additionally, the rate of ATP hydrolysis and the protease activity were also not affected by the inhibitors. Thus, these results indicate that the three ITBA analogs containing N-naphthoyl moieties are the foundation of a potential series of small molecules capable of inhibiting NS3 activity via a novel interaction with the helicase domain that prevents the productive unwinding of nucleic acid substrates, and may represent the basis for a new class of therapeutic agents with the potential to aid in the treatment and eradication of hepatitis C virus.

Arm lymphedema is a major complication after surgery and/or radiation for breast cancer. This life‐long and disfiguring complication can cause serious medical problems including lymphangitis, cellulitis, and other recurrent infections. Doxorubicin ( DOX ) is often a central component in chemotherapy to prevent metastases or recurrence of breast cancer, but this cytotoxic drug increases the incidence and severity of lymphedema as an off‐target effect. Interestingly, the mechanisms by which DOX contributes to arm lymphedema have not been elucidated. We hypothesized that DOX directly suppresses lymphatic drainage by disrupting lymph vessel (LV) contractile function. Rats were anesthetized and one loop of mesentery was exposed on a heated microscope stage. High‐resolution optical imaging of rat mesenteric LVs in vivo revealed that continuous superfusion of DOX (0.05 to 10 μmol/L) progressively reduced positive volumetric lymph flow to ~30% of baseline (t=20 min, n=6) with an IC 50 of 0.66 μmol/L. This effect of DOX was partially reversed by drug washout. Data were corrected for solvent and time controls (n=5–6). In a subsequent study, baseline measurements were recorded for 10 min, and then rats were injected via tail vein with a clinically relevant dose of DOX (10 mg/kg) or equivalent volume of saline. Mesenteric lymph flow was measured continuously for 2 hr post‐injection. At 0.75–1.25 hr post‐ injection, DOX significantly reduced positive volumetric flow to ~45% of baseline, whereas saline injection increased positive volumetric flow by ~35% from baseline (n=4–5). HPLC was used to determine corresponding DOX plasma levels during the measurement period. The average plasma concentration of DOX was 0.15±0.06 μmol/L (86 ± 34 ng/mL) and 0.10±0.02 μmol/L (59±13 ng/mL) at 1 and 2 hr after injection, respectively (n=5). These values were similar to DOX concentrations used in the superfusate studies and equivalent to plasma DOX concentrations achieved during chemotherapy in patients. Thus, clinically relevant concentrations of DOX appear to directly and acutely inhibit LV contractile function in vivo , and the resulting compromise of lymph flow represents a possible contributing mechanism of drug‐induced lymphedema. Support or Funding Information Funded by Rho Chi‐AFPE Graduate Fellowship, UAMS Pilot Grant, and NIH/NCI R21CA131164 Grant.

Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to “DNA sensors,” which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.

SIGNIFICANCE:Respiring mitochondria are a significant site for reactions involving reactive oxygen and nitrogen species that contribute to irreversible cellular, structural, and functional damage leading to multiple pathological conditions. Manganese superoxide dismutase (MnSOD) is a critical component of the antioxidant system tasked with protecting the oxidant-sensitive mitochondrial compartment from oxidative stress. Since global knockout of MnSOD results in significant cardiac and neuronal damage leading to early postnatal lethality, this approach has limited use for studying the mechanisms of oxidant stress and the development of disease in specific tissues lacking MnSOD. To circumvent this problem, a number of investigators have employed the Cre/loxP system to precisely knockout MnSOD in individual tissues.RECENT ADVANCES:Multiple tissue and organ-specific Cre-expressing mice have been generated, which greatly enhance the specificity of MnSOD knockout in tissues and organ systems that were once difficult, if not impossible to study.CRITICAL ISSUES:Evaluating the contribution of MnSOD deficiency to oxidant-mediated mitochondrial damage requires careful consideration of the promoter system used for creating the tissue-specific knockout animal, in addition to the collection and interpretation of multiple indices of oxidative stress and damage.FUTURE DIRECTIONS:Expanded use of well-characterized tissue-specific promoter elements and inducible systems to drive the Cre/loxP recombinational events will lead to a spectrum of MnSOD tissue knockout models, and a clearer understanding of the role of MnSOD in preventing mitochondrial dysfunction in human disease.